The present invention relates to the control of gene expression. More particularly, this invention relates to the use of synthetic, modified oligonucleotides to down-regulate the expression of a gene in an animal.
The potential for the development of an antisense oligonucleotide therapeutic approach was first suggested in three articles published in 1977 and 1978. Paterson et al. (Proc. Natl. Acad Sci. (USA) (1977) 74:4370-4374) discloses that cell-free translation of mRNA can be inhibited by the binding of an oligonucleotide complementary to the mRNA. Zamecnik et al. (Proc. Natl. Acad. Sci. (USA) (1978) 75:280-284 and 285-288) discloses that a 13mer synthetic oligonucleotide that is complementary to a part of the Rous sarcoma virus (RSV) genome inhibits RSV replication in infected chicken fibroblasts and inhibits RSV-mediated transformation of primary chick fibroblasts into malignant sarcoma cells.
These early indications that synthetic oligonucleotides can be used to inhibit virus propagation and neoplasia have been followed by the use of synthetic oligonucleotides to inhibit a wide variety of viruses, such as HIV (see, e.g., U.S. Pat. No. 4,806,463); influenza (see, e.g., Leiter et al. (1990) (Proc. Natl. Acad. Sci. (USA) 87:3430-3434); vesicular stomatitis virus (see, e.g., Agris et al. (1986) Biochem. 25:6268-6275); herpes simplex (see, e.g., Gao et al. (1990) Antimicrob. Agents Chem. 34:808-812); SV40 (see, e.g., Birg et al. (1990) (Nucleic Acids Res. 18:2901-2908); and human papilloma virus (see, e.g., Storey et al. (1991) (Nucleic Acids Res. 19:4109-4114). The use of synthetic oligonucleotides and their analogs as antiviral agents has recently been extensively reviewed by Agrawal (Trends in Biotech. (1992) 10:152-158).
In addition, synthetic oligonucleotides have been used to inhibit a variety of non-viral pathogens, as well as to selectively inhibit the expression of certain cellular genes. Thus, the utility of synthetic oligonucleotides as agents to inhibit virus propagation, propagation of non-viral, pathogens and selective expression of cellular genes has been well established.
Improved oligonucleotides have more recently been developed that have greater efficacy in inhibiting such viruses, pathogens and selective gene expression. Some of these oligonucleotides having modifications in their internucleotide linkages have been shown to be more effective than their unmodified counterparts. For example, Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1988) 85:7079-7083) teaches that oligonucleotide phosphorothioates and certain oligonucleotide phosphoramidates are more effective at inhibiting HIV-1 than conventional phosphodiester-linked oligodeoxynucleotides. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1989) 86:7790-7794) discloses the advantage of oligonucleotide phosphorothioates in inhibiting HIV-1 in early and chronically infected cells.
In addition, chimeric oligonucleotides having more than one type of internucleotide linkage within the oligonucleotide have been developed. Pederson et al. (U.S. Pat. Nos. 5,149,797 and 5,220,007 discloses chimeric oligonucleotides having an oligonucleotide phosphodiester or oligonucleotide phosphorothioate core sequence flanked by nucleotide methylphosphonates or phosphoramidates. Furdon et al. (Nucleci Acids Res. (1989) 17:9193-9204) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters in addition to either oligonucleotide phosphorothioate or methylphosphonate regions. Quartin et al. (Nucleic Acids Res. (1989) 17:7523-7562) discloses chimeric oligonucleotides having regions of oligonucleotide phosphodiesters and oligonucleotide methylphosphonates. Inoue et al. (FEBS Lett. (1987) 215:237-250) discloses chimeric oligonucleotides having regions of deoxyribonucleotides and 2xe2x80x2-O-methyl-ribonucleotides.
Many of these modified oligonucleotides have contributed to improving the potential efficacy of the antisense oligonucleotide therapeutic approach. However, certain deficiencies remain in the known oligonucleotides, and these deficiencies can limit the effectiveness of such oligonucleotides as therapeutic agents. For example, Wickstrom (J. Biochem Biophys. Meth. (1986) 13:97-102) teaches that oligonucleotide phosphodiesters are susceptible to nuclease-mediated degradation, thereby limiting their bioavailability in vivo. Agrawal et al. (Proc. Natl. Acad. Sci. (USA) (1990) 87:1401-1405) teaches that oligonucleotide phosphoramidates or methylphosphonates when hybridized to RNA do not activate RNase H, the activation of which can be important to the function of antisense oligonucleotides. Thus, a need for methods of controlling gene expression exists which uses oligonucleotides with improved therapeutic characteristics.
Several reports have been published on the development of phosphorothioate-linked oligonucleotides as potential anti-AIDS therapeutic agents. Although extensive studies on chemical and molecular mechanisms of oligonucleotides have demonstrated the potential value of this novel therapeutic strategy, little is known about the pharmacokinetics and metabolism of these compounds in vivo.
Several preliminary studies on this topic have been published. Agrawal et al. (Proc. Natl. Acad. Sci.(USA) (1991) 88:7595-7599) describes the intravenous and intraperitoneal administration to mice of a 20mer phosphorothioate linked-oligonucleotide. In this study, approximately 30% of the administered dose was excreted in the urine over the first 24 hours with accumulation preferentially in the liver and kidney. Plasma half-lives ranged from about 1 hour (t1/2xcex1) and 40 hours (t1/2xcex2), respectively. Similar results have been reported in subsequent studies (Iversen (1991) Anti-Cancer Drug Design 6:531-538; Iversen (1994) Antisense Res. Devel. 4:43-52; and Sands (1994) Mol.Phann. 45:932-943). However, stability problems may exist when oligonucleotides are administered intravenously and intraperitoneally. More recently, Agrawal et al. reported that oligonucleotide hybrids containing 2xe2x80x2-O-methyl ribonucleotides at both the 3xe2x80x2- and 5xe2x80x2 ends and deoxyribonucleotide phosphorothioates in the interior portion were absorbed through the gastrointestinal (GI) tract of rats (Biochem. Pharm. (1995) 50:571-576).
Thus, there remains a need to develop more effective therapeutic methods of down-regulating the expression of genes which can be easily manipulated to fit the animal and condition to be treated, and the gene to be targeted. Preferably, these methods should be simple, painless, and precise in effecting the target gene.
The present invention provides a method of down-regulating the expression of a gene in an animal which involves the administration of an oligonucleotide complementary to the gene via an oral route, thereby bypassing the complications which may be experienced during intravenous and other modes of in vivo administration.
It has been discovered that hybrid oligonucleotides with other than phosphodiester bonds and having at least one 2xe2x80x2-substituted ribonucleotide and chimeric oligonucleotides with at least two different types of internucleotide linkages are relatively stable in vivo following oral administration to an animal, and that these molecules are successfully absorbed from the gastrointestinal tract and distributed to various body tissues. This discovery has been exploited to develop the present invention, which is a method of down-regulating the expression of a gene in an animal.
This method is also a means of examining the function of various genes in an animal, including those essential to animal development. Presently, gene function can only be examined by the arduous task of making a xe2x80x9cknockoutxe2x80x9d animal such as a mouse. This task is difficult, time-consuming and cannot be accomplished for genes essential to animal development since the xe2x80x9cknock outxe2x80x9d would produce a lethal phenotype. The present invention overcomes the shortcomings of this model.
In the method of the invention, a pharmaceutical formulation containing an oligonucleotide complementary to the targeted gene is orally administered in a pharmaceutically acceptable carrier to the animal harboring the gene. The oligonucleotide inhibits the expression of the gene, thereby down-regulating its expression.
For purposes of the invention, the term xe2x80x9canimalxe2x80x9d is meant to encompass humans as well as other mammals, as well as reptiles amphibians, and insects. The term xe2x80x9coral administrationxe2x80x9d refers to the provision of the formulation via the mouth through ingestion, or via some other part of the gastrointestinal system including the esophagus.
As used herein, the term xe2x80x9coligonucleotidexe2x80x9d is meant to include polymers of two or more nucleotides or nucleotide analogs connected together via 5xe2x80x2 to 3xe2x80x2 internucleotide linkages which may include any linkages that are known in the antisense art, including non-phosphodiester linkages. Such molecules have a 3xe2x80x2 terminus and a 5xe2x80x2 terminus.
The term xe2x80x9cnon-phosphodiester-linkagesxe2x80x9d as used herein refers to a synthetic covalent attachment between the 5xe2x80x2 end of one nucleotide and the 3xe2x80x2 end of another nucleotide in which the 5xe2x80x2 nucleotide phosphate has been replaced with any number of chemical groups. Preferable synthetic linkages include alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, phosphoramidates, phosphoramidites, phosphate esters, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. In one preferred embodiment of the invention, all of the nucleotides of the oligonucleotide are linked via phosphorothioate and/or phosphorodithioate linkages.
In some embodiments of the invention, the oligonucleotides administered are modified with other than, or in addition to, non-phosphodiester-internucleotide linkages. As used herein, the term xe2x80x9cmodified oligonucleotidexe2x80x9d encompasses oligonucleotides with modified nucleic acid(s), base(s), and/or sugar(s) other than those found in nature. For example, a 3xe2x80x2, 5xe2x80x2-substituted oligonucleotide is an oligonucleotide having a sugar which, at both its 3xe2x80x2 and 5xe2x80x2 positions is attached to a chemical group other than a hydroxyl group (at its 3xe2x80x2 position) and other than a phosphate group (at its 5xe2x80x2 position).
A modified oligonucleotide may also be one with added substituents such as diamines, cholestryl, or other lipophilic groups, or a capped species. In addition, unoxidized or partially oxidized oligonucleotides having a substitution in one nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides. Also considered as modified oligonucleotides are oligonucleotides having nuclease resistance-conferring bulky substituents at their 3xe2x80x2 and/or 5xe2x80x2 end(s) and/or various other structural modifications not found in vivo without human intervention are also considered herein as modified.
In one embodiment, the oligonucleotide being administered in the method of the invention has non-phosphodiester internucleotide linkages and includes at least one 2xe2x80x2-substituted ribonucleotide.
For purposes of the invention, the term xe2x80x9c2xe2x80x2-substituted oligonucleotidexe2x80x9d refers to an oligonucleotide having a sugar attached to a chemical group other than a hydroxyl group at its 2xe2x80x2 position. The 2xe2x80x2-OH of the ribose molecule can be substituted with -O-lower alkyl containing 1-6 carbon atoms, aryl or substituted aryl or allyl having 2-6 carbon atoms, e.g., 2xe2x80x2-O-allyl, 2xe2x80x2-O-aryl, 2xe2x80x2-O-alkyl (such as a 2xe2x80x2-O-methyl), 2xe2x80x2-halo, or 2xe2x80x2-amino, but not with 2xe2x80x2-H, wherein allyl, aryl, or alkyl groups may be unsubstituted or substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl or amino groups.
In one preferred embodiment of the invention, the oligonucleotide administered includes at least one 2xe2x80x2-substituted ribonucleotide at its 3xe2x80x2 terminus. In some embodiments, all but four or five nucleotides at its 5xe2x80x2 terminus are 2xe2x80x2-substituted ribonucleotides, and in some embodiments, these four or five unsubstituted 5xe2x80x2 nucleotides are deoxyribonucleotides. In other embodiments, the oligonucleotide has at least one 2xe2x80x2-substituted ribonucleotide at both its 3xe2x80x2 and 5xe2x80x2 termini, and in yet other embodiments, the oligonucleotide is composed of 2xe2x80x2-substituted ribonucleotides in all positions with the exception of at least four or five contiguous deoxyribonucleotide nucleotides in any interior position. Another aspect of the invention includes the administration of an oligonucleotide composed of nucleotides that are all 2xe2x80x2-substituted ribonucleotides. Particular embodiments include oligonucleotides having a 2xe2x80x2-O-alkyl-ribonucleotide such as a 2xe2x80x2-O-methyl. Other embodiments include the administration of chimeric oligonucleotides. In one preferred embodiment, the chimeric oligonucleotide has at least one alkylphosphonate internucleotide linkage at both its 3xe2x80x2 and 5xe2x80x2 ends and having phosphorothioate internucleotide linkages.
In another embodiment of the invention, the oligonucleotide administered has at least one deoxyribonucleotide, and in a preferred embodiment, the oligonucleotide has at least four or five contiguous deoxyribonucleotides capable of activating RNase H.
The oligonucleotide administered is complementary to a gene of a virus, pathogenic organism, or a cellular gene in some embodiments of the invention. In some embodiments, the oligonucleotide is complementary to a gene of a virus involved in AIDS, oral or genital herpes, papilloma warts, influenza, foot and mouth disease, yellow fever, chicken pox, shingles, adult T-cell leukemia, Burkitt""s lymphoma, nasopharyngeal carcinoma, or hepatitis. In one particular embodiment, the oligonucleotide is complementary to an HIV gene and includes about 15 to 26 nucleotides linked by phosphorothioate internucleotide linkages, at least one of the nucleotides at the 3xe2x80x2 terminus being a 2xe2x80x2-substituted ribonucleotide, and at least four contiguous deoxyribonucleotides.
In another embodiment, the oligonucleotide is complementary to a gene encoding a protein associated with Alzheimer""s disease.
In yet other embodiments, the oligonucleotide is complementary to a gene encoding a protein expressed in a parasite that causes a parasitic disease such as amebiasis, Chagas"" disease, toxoplasmosis, pneumocytosis, giardiasis, cryptoporidiosis, trichomoniasis, malaria, ascariasis, filariasis, trichinosis, or schistosomiasis infections.